1. Field of the Invention
The present invention relates to a method for determination of direct bilirubin contained in a sample of body fluid and a reagent for use in the method.
2. Related Art
Bilirubin is a metabolic product of hemoglobin derived from aged erythrocyte and is the main component of bile pigment. Blood bilirubin contains as predominant components direct bilirubin (conjugate form) and indirect bilirubin (free form). Direct bilirubin having propionic acid groups on the side chain, which enzymatically forms an ester bond mainly with glucuronic acid in the liver, is highly water soluble, and reacts readily with a diazo reagent to form an azo dye substance. Indirect bilirubin, the propionic acid groups of which are in a free state, has a low water solubility and reacts with a diazo reagent only in the presence of a reaction accelerator such as an alcohol, etc. Indirect bilirubin can be determined by subtracting direct bilirubin from total bilirubin which is a measurement value of conjugated form and free form in total, obtained by a diazo reaction with a diazo reagent in the presence of a reaction accelerator.
Individual bilirubin concentrations of conjugate (direct) form and free (indirect) form can thus be separately determined to make diagnosis of various liver diseases and diacrisis of jaundice. Therefore, the measurement of bilirubin is one of important clinical tests.
There have been proposed methods for quantitative determination of direct bilirubin as described below, such as a method using a diazo reagent, a method using bilirubin oxidase, a method using high performance liquid chromatography, a method using a chemical oxidizing substance, and the like.
A) Assay for direct bilirubin using a diazo reagent
In the diazo method, bilirubin reacts with a diazo reagent to form azobilirubin. The azobilirubin has an absorption maximum which is higher than the visible absorption maximum of bilirubin itself and is easily detectable by optical changes. The method using a diazo reagent has many variations due to a kind of a reaction accelerator for indirect bilirubin, conditions for reaction termination and conditions for detecting azobilirubin (Malloy, H. T., Evelyn, K. A.: J. Biol. Chem., 119, 481 (1937); The determination of bilirubin with the photoelectric calorimeter; Jendrassik, L., Grof, P., Biochem. Z., 297, 81 (1938): Vereinfachte Photometrische Methoden zur Bestimmung des Blutbilirubins; Micha elsson, M., Scand. J. Clin. Lab. Invest., 12 (Supp. 56), 1-80 (1937): Bilirubin determination in serum and urine).
B) Assay for direct bilirubin using bilirubin oxidase
In the method using a bilirubin oxidase, the enzyme is acted on a sample suspected of containing bilirubin to oxidize bilirubin to biliverdin, whereupon absorbance of bilirubin disappears in the maximum absorption wavelength region. Direct bilirubin can thus be determined by this decrease in the absorbance. Various modifications have been made to inhibit a reaction of indirect bilirubin. The following methods are proposed for such modifications.
B1) Method for determination of direct bilirubin in which a bilirubin oxidase is reacted in a pH range of 3.5 to 4.5 (Japanese Patent KOKAI (Laid-Open) No. 59-125899);
B2) Method for determination of direct bilirubin which involves reacting a bilirubin oxidase with bilirubin in an acidic buffer solution of pH 5 to 6 containing an anionic surfactant (Shogo Otsuji: Clin. Biochem., 21, 33-38 (1988) and Japanese Patent KOKAI (Laid-Open) No. 60-152955);
B3) Method for quantitative determination of conjugated bilirubin which involves reacting a bilirubin oxidase in a buffer solution of pH 9 to 10 and measuring a change in absorbance (Japanese Patent KOKAI (Laid-Open) No. 62-58999);
B4) Method for quantitative determination of direct bilirubin which involves reacting a bilirubin oxidase in a buffer solution of pH 2.0 to 3.3 containing potassium ferrocyanide and/or potassium ferricyanide and measuring a change in absorbance (Japanese Patent KOKAI (Laid-Open) No. 64-5499);
B5) Method for quantitative determination of direct bilirubin which involves reacting a bilirubin oxidase in the presence of a fluorine compound or a reducing agent (Japanese Patent KOKAI (Laid-Open) No. 5-276992); and
B6) Method for quantitative determination of direct bilirubin which involves allowing to act a bilirubin oxidase in the presence of a tetrapyrrole compound (Japanese Patent KOKAI (Laid-Open) No. 7-231795).
C) Method for determination of direct bilirubin using high performance liquid chromatography (HPLC)
The method using HPLC involves elution of bilirubin by an organic solvent gradient on a reversed phase column and fractionation of the bilirubin fractions due to difference in the hydrophilic/hydrophobic property. According to HPLC, serum bilirubin is fractionated into 4 fractions of .alpha., .beta., .gamma. and .delta.. The .alpha., .beta., .gamma. and .delta. fractions are identified, respectively, to free form of bilirubin, bilirubin in which only one of the two propionic acid groups on the side chain in one molecule forms an ester bond with glucuronic acid (bilirubin monoglucuronide), bilirubin in which the two propionic acid groups form ester bonds with glucuronic acid (bilirubin diglucuronide) and bilirubin which forms covalent bond to albumin. The .delta. fraction is assumed to be formed by non-enzymatic reaction of the .gamma. fraction with albumin (Toshio Yamamoto, Nippon Naibunpi Gakkai Zasshi, 78 (11), 36-41 (1989)). It is recognized that the .alpha. fraction obtained by HPLC corresponds to indirect bilirubin and the .beta. and .gamma. fractions correspond to direct bilirubin, when determined by the method using a diazo reagent (John J. Lauff, Clin. Chem., 28 (4) 629-637 (1982)). The method using HPLC has been continuously modified to improve complicated pretreatment of a sample and such modifications are reported in Nakamura, H.: Bunseki Kagaku, 36, 352-355 (1987); Yukihiko Adachi: Gastroenterologia Japonica, 23 (3), 268-272 (1988); Yuko Kato: Kinkidaigaku Igaku Zasshi, 14 (1), 97-112 (1989).
D) Assay for direct bilirubin using a chemical oxidizing agent
The method using a chemical oxidizing agent involves reacting a low molecular oxidizing agent in lieu of a bilirubin oxidase to oxidize bilirubin to biliverdin. When oxidized, the absorbance of bilirubin decreases. Accordingly, direct bilirubin can be determined based on the decreased absorbance. Various modifications have been also made in this method for inhibiting a reaction of indirect bilirubin. The following methods are proposed for such modifications.
D1) Method for quantitative determination of direct bilirubin characterized in that copper ions and thiourea or a derivative thereof are reacted with a sample solution (Japanese Patent KOKAI (Laid-Open) No. 63-118662).
D2) Method for quantitative determination of bilirubin characterized in that vanadic acid ions or trivalent manganese ions are reacted as an oxidizing agent to measure optical changes of a sample solution (Japanese Patent KOKAI (Laid-Open) No. 5-18978). In order to measure direct bilirubin by this method, one or more compounds selected from the group consisting of hydrazines, hydroxylamines, oximes, aliphatic polyvalent amines, phenols, water soluble high molecular weight substances and non-ionic surfactants having an HLB of at least 15 is/are employed as a reaction inhibitor for indirect bilirubin.
D3) Method for quantitative determination of bilirubin characterized in that nitrous acid is reacted as an oxidizing agent to measure optical changes of a sample solution (WO 96-17251). To effect the measurement of the direct bilirubin by this method, a reaction inhibitor for indirect bilirubin such as polyoxyethylene (n-alkyl or iso-alkyl) ether having an HLB of 12 to 15, thiourea, hydrazine, polyvinylpyrrolidone, or the like is employed.
Every one of these methods A) to D) has both advantageous and disadvantages and none of them has been completely satisfactory for the bilirubin assay required. The disadvantages involved in these methods are described below.
In the method A) using a diazo reagent, a reaction in the absence of any reaction accelerator is called a diazo direct reaction, from which the term direct bilirubin originates. However, it has been reported in many journals that a part of indirect bilirubin could also cause this diazo direct reaction (e.g., Killenberg, P. G., Gastroenterology, 78, 1011-1015 (1980); Blankaert, N., J. Lab. Clin. Med., 96, 198-212 (1980); Yukio Manabe, BUNSEKI KAGAKU, 30, 736-740 (1981); Chan, K. M., Clin. Chem., 31, 1560-1563 (1985); Akira Kosaka, KENSA-TO-GIJUTSU, 14, 971-975 (1986); Yukihiko Adachi, SEIBUTSU-SHIRYOU BUNSEKI, 9, 33-42 (1986)). Therefore, even though bilirubin is determined by the so-called diazo direct method, the thus obtained bilirubin value does not strictly represent "direct bilirubin".
The method B) using a bilirubin oxidase has been developed to obtain bilirubin values as close to the measurement values defined by the direct diazo reaction as possible. As a natural consequence, oxidation is also caused partly with indirect bilirubin and hence basically, the method B) does not give precise measurement of "direct bilirubin". This has necessitated modifications of the method B) to avoid any undesirable reaction with indirect bilirubin. In an improved method, a bilirubin oxidase is allowed to act in the presence of a fluorine compound (Japanese Patent KOKAI (Laid-Open) No. 5-276992) and in another method a bilirubin oxidase is allowed to act in the presence of a tetrapyrrole compound (Japanese Patent KOKAI (Laid-Open) No. 7-231795). However, these methods encounter problems that use of a fluorine compound results in environmental pollution in the former and the necessity for the tetrapyrrole compound to be present in a reagent solution makes the solution unstable which does not enable to use the assay system in a solution state for time required for the assay.
The method C) using HPLC provides a high efficiency of analysis but on the other hand requires a considerably long time for about an hour to treat one sample. Therefore, the method C) is inadequate for treating a large number of samples. Moreover, the method C) requiring expensive and special apparatuses is not available for all purposes.
The method D) using a chemical oxidizing agent involves similar problems as observed in the method B) using a bilirubin oxidase, since the method D) has also been developed to obtain bilirubin measurement values as close to those defined by the direct diazo reaction as possible and hence, a part of indirect bilirubin is oxidized. In this regard, it is difficult to say that the method D) precisely measures "direct bilirubin".
As stated above, none of the foregoing methods can perfectly avoid the interference of indirect bilirubin or can provide stable and safe measurement of direct bilirubin. Therefore, it has been strongly desired to develop a method for determination of direct bilirubin deprived of these defects in the prior art and satisfying all the requirements.